Development of an ELISA for myeloperoxidase on microplate: Normal reference values and effect of temperature on specimen preparation

BackgroundMyeloperoxidase (MPO), a leukocyte enzyme, is implicated in both the pathogenesis and the progression of atherosclerosis.MethodsWe developed a sandwich ELISA on microplate using commercial antibodies for the measurement of plasma MPO.ResultsThe in-house ELISA has a sensitivity of 15 ng/ml. Both within-day and between-day imprecision were < 10%. The in-house assay was well correlated with the commercial kit from Oxis (γ = 0.96). We have established normal reference range for MPO for apparently healthy Chinese. No statistical difference was found between males and females and the various age groups. Because the coating antibodies used by two different kits are different in their affinities for MPO, the analysis by the in-house ELISA that was approximately three times that of the Oxis kit when testing the same specimens. We found that it is necessary to keep the heparinized whole blood on ice before centrifugation in order to prevent further release of MPO from the leukocytes at room temperature. For the same reason, serum is not recommended for MPO measurement. We also found that either pooled human plasma or serum containing MPO can be used as calibrators.ConclusionsWe believe that this ELISA for MPO is useful to assess risk for inflammation, oxidative stress, nitrosative stress, and for predicting cardiac events.